Theobroma cacao L. is a tropical tree originating in the Amazon, where it occurred naturally in the shade of the tropical rainforests. Cacao sub-products like butter and powder are produced as principal components of chocolate, and contain with important compounds for nutrition as polyphenols and flavonoids, among others. However, bean production is decreasing due to the fact that many plantations are old and unproductive. Propagation of cacao has been traditionally carried out through classical propagation methods, such as grafting or rooted-cuttings, but those are not sufficient for obtaining large amounts of planting material with the desired genetic quality and plant health.
In the search for solutions to this problem, somatic embryogenesis is one of the vegetative methods for propagation of cacao that shows potential for providing large quantities of high-quality planting material. Somatic embryos are formed from plant cells that are not normally involved in the development of embryos, i.e., ordinary plant tissue. Applications of the somatic embryogenesis process include: clonal propagation of genetically uniform plant material; elimination of viruses; provision of source tissue for genetic transformation; generation of whole plants from single cells; and development of synthetic seed technology.
Somatic embryogenesis is a type of clonal propagation where competent cells in the somatic tissue can develop into embryos and subsequently convert into plants. In indirect somatic embryogenesis, cells derived from competent source tissue can be cultured to form an undifferentiated mass of cells called a callus. Plant growth regulators in the tissue culture medium can be manipulated to induce callus formation and subsequently changed to induce embryos to form from the callus. The ratio of different plant growth regulators required to induce callus or embryo formation varies with the type of plant.
This method offers significant technological advantages, because it is possible to obtain a large amount of disease-free planting material with good agronomic characteristics and genetic stability. However, tissue culture techniques can be cumbersome and there is significant variation between the protocols and conditions that are required to produce different plant types or species.
The majority of the work to date in micropropagation of cacao through somatic embryogenesis uses the technique of indirect embryogenesis (Maximova et al., 2002 In Vitro Cell Dev. Biol Plant 38:252-259 and WO 2009/071254) in which undifferentiated calluses are obtained as a source of primary embryos.
WO 2009/071254 describes Theobroma cacao L. propagation by indirect somatic embryogenesis in solid or liquid media cocoa flower buds and leaves which are subjected to:                i) primary embryogenesis in the dark in a solid culture medium for 5 to 15 weeks for causing induction and expression to produce primary embryos,        ii) secondary embryogenesis                    (a) in which the primary embryos are treated in the dark in solid or liquid media culture medium for 10 to 25 weeks to produce and multiply embryogenesis callus followed by            (b) treatment of the embryogenesis callus in the dark in a suitable liquid culture medium for 1 to 6 weeks for causing expression of the embryogenesis callus to produce further new secondary embryos,                        iii) pre-germination of the secondary embryos in a Petri dish on a solid medium, or in a bioreactor in a liquid medium for 3 to 12 weeks, into pre-germinated secondary embryos at the cotyledonary stage,        iv) ex vitro germination of the pre-germinated secondary embryos at the cotyledonary stage by sowing directly on a culture substrate in the greenhouse to produce the plantlets, and        v) development of the plantlets.        
However, such systems require a long period to obtain somatic embryos and regenerated plants, and can result in a high amount of abnormal embryos. Furthermore, because of the reliance on formation of callus tissue, there is a higher probability of somaclonal variation among the regenerated plants.
In the approach of direct embryogenesis, somatic embryos may be derived from plant tissues comprising differentiated cells. The differentiated cells may include specialized cells such as the differentiated cells of cicatricial callus that forms on the epidermis of a plant, for example, in response to a wound. The differentiated cells of the explant material used in direct somatic embryogenesis is therefore different from the undifferentiated callus used in indirect somatic embryogenesis. The method comprises the cultivation of explants in culture media supplemented only with cytokinin, or the combination of auxin and cytokinin (Dublin, Café Cacao Thé, Paris, 25(4), 237-241, 1981; Pierson et al., Protoplasma 115, 208-216, 1983). Some authors, such as Dublin (1981), suggest that propagation by direct somatic embryogenesis is suitable for keeping the stability of the donator genotype in some species, such as coffee.
Typically processes based upon direct somatic embryogenesis involve the following sequence of steps:
i) Induction of direct somatic embryogenesis
ii) Development of somatic embryos
iii) Multiplication of direct somatic embryos
iv) Germination of somatic embryos
v) Conversion in plants of somatic embryos.
However, previous attempts to apply this technique to the propagation of cacao have not been satisfactory. For example, it was reported by Litz, R. E. (“Tissue Culture Studies with T. cacao,” Cacao Biotechnology Symposium, Pennsylvania State University, U.S.A. 1986) that a low frequency of somatic embryogenesis from young leaves was obtained using a semi-solid MS medium (Murashige and Skoog, 1962), supplemented with sucrose, and high levels of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the synthetic cytokinin 6-benzylaminopurine (BAP). The somatic embryos developed only to the heart stage. Some direct embryogenesis was observed at the base of young petal explants from immature flowers of cacao after three weeks of culture (Sondahl, M. R. et al. “Somatic Embryogenesis and Plan Regeneration of cacao.” Acta Hort. Republic of South África. 1993), when grown in liquid medium with different combinations of BAP, indole-3-acetic acid (IAA), gibberellic acid (GA3), abscisic acid (ABA), and 2,4-D, supplemented with coconut water and kept in the dark. The response was dependent on genotype.
U.S. Pat. No. 6,197,587 relates to methods of (i) inducing indirect somatic embryogenesis from cacao tissue explants, and (ii) regenerating cacao plants from somatic embryos. Tissue culture media adapted for use in the methods are provided, including primary callus growth medium, secondary callus growth medium, embryo development medium, primary embryo conversion medium, secondary embryo conversion medium, and plant regeneration medium. Although the process of inducing somatic embryos in cacao explants may be carried out in the dark or under light, darkness is preferred.
U.S. Pat. No. 5,312,801 relates to a method for regeneration of somatic embryos from non-zygotic or zygotic tissue, and provides a means for regeneration of cacao plantlets and plants from somatic embryos. Although it is indicated that somatic embryos may be derived from any source, the focus is on the use of nucellus and young flower bud petals. The methods are carried out in low light (300-100 lux, which is equivalent to 5.76-1.95 μmol/m2/sec) or in darkness.
DE 102010044562 A1 relates to automated techniques for in vitro propagation of plants based on somatic embryogenesis, and a process for forming homogenous plant clusters based on a regeneration technique using stem cells from the apical meristem. The resulting directed proliferation under targeted expression of phytohormones leads to formation of homogenous secondary embryoids with unlimited proliferation ability. A nutrient medium having a composition that is adjusted to induce defined differentiation is provided for controllable plant regeneration. The methods are carried out using small explants taken from meristematic material.
The article by Vinterhalter relates to direct somatic embryogenesis in celandine (Chelidonium majus L.) using whole epicotyls explants of seedlings after prolonged cultivation on MS medium, with or without plant growth regulators. Somatic embryos developed into plantlets, which entered additional cycles of somatic embryogenesis. Embryos that developed into rooted plantlets could be acclimated in a greenhouse.
The article by Iantcheva relates to a procedure for direct somatic embryogenesis from wild Medicago spp. using various explants, including meristematic zones (hypocotyls, cotyledons, and petiole bases from seeds). Solid medium supplemented with thidiazuron or 6-benzylaminopurine at different concentrations promoted the process. Secondary embryogenesis was also observed. Cuttings of clusters of primary and secondary embryos were used for cyclic production of new embryo generations. Regenerated plants with well-developed root systems on medium with reduced levels of macroelements and sucrose were adapted to a greenhouse.
The article by Guiltinan relates to a procedure for the stimulation of somatic embryogenesis and plant regeneration from non-zygotic somatic tissues of cacao, involving the use of several culture steps in combination with the use of the synthetic cytokinin thidiazuron (TDZ) and a synthetic auxin (2,4D). Three steps are used, including callus induction, embryo development, and plant regeneration. When used in the process, 100% of explants taken from staminodes and 60% of explants taken from petal bases produced somatic embryos, and up to 37% of the selected mature somatic embryos produced were capable of conversion into plantlets.
In contrast to the indirect somatic embryogenesis methods described in the prior art, the present invention beneficially provides methods for producing embryos using techniques that include direct somatic embryogenesis. Accordingly, the methods of the invention beneficially provide a unique opportunity for tree breeders and cocoa researchers to improve genetic uniformity in plants for use in further testing, and/or improve yields and disease resistance in trees grown for use in cocoa production.